Inhalable aztreonam for treatment and prevention of pulmonary bacterial infections

ABSTRACT

A method and a composition for treatment of pulmonary bacterial infections caused by gram-negative bacteria suitable for treatment of infection caused by  Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Haemophilus influenzae, Proteus mirabilis, Enterobacter  species,  Serratia marcescens  as well as those caused by  Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans,  and multidrug resistant  Pseudomonas aeruginosa,  using a concentrated formulation of aztreonam, or a pharmaceutically acceptable salt thereof, delivered as an aerosol or dry powder formulation.

This application is a Continuation of the U.S. application Ser. No.10/654,815, filed on Sep. 4, 2003, allowed, which is a Divisional of theU.S. application Ser. No. 10/027,113, filed on Dec. 20, 2001, issued onDec. 9, 2003 as the U.S. Pat. No. 6,660,249, that claims priority of theProvisional application Ser. No. 60/258,423, filed on Dec. 27, 2000,incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

2. Background and Related Disclosures

A wide variety of gram-negative bacteria cause severe pulmonaryinfections. Many of these bacteria are or become resistant to commonlyused or specialty antibiotics and require treatment with new types ofantibiotics. The pulmonary infections caused by gram-negative bacteriaare particularly dangerous to patients who have decreasedimmunoprotective responses, such as for example cystic fibrosis and HIVpatients, patients with bronchiectasis or those on mechanicalventilation.

Therefore, the bacterial respiratory infections caused by organismsresistant to antibiotics continues to be a major problem, particularlyin immunocompromised or hospitalized patients, as well as in patientsassisted by mechanical ventilation, as described in Principles andPractice of Infectious Diseases, Eds. Mandel, G. L., Bennett, J. E., andDolin, R., Churchill Livingstone Inc., New York, N.Y., (1995).

Currently accepted therapy for severe bacterial respiratory tractinfections, particularly for treatment of pneumonia in patients withunderlying illnesses, includes treatment with various intravenousantibacterial agents, often used in two or three way combination. Mostof these agents are not suitable, available or FDA approved for eitheroral or aerosol dosing. In some cases the efficacious systemicintravenous or oral dose, if oral delivery is possible, requires doseswhich are borderline or outright toxic thus often preventing a use ofperfectly good antibiotic for treatment of the pulmonary infections.

Thus it would be desirable to have available other modes of deliveryroutes of these antibiotics enabling a targeted delivery of smalleramounts of the antibiotic to endobronchial space of airways fortreatment of these bacterial infections rather than administering theantibiotic systemically in large amounts.

Additionally, chronically ill patients are often affected withinfections caused by bacteria which are largely resistant to commonlyused antibiotics or, upon extended use of certain antibiotic, oftendevelop strong resistance to such antibiotic. For example, chronicpulmonary colonization with Pseudomonas aeruginosa in patients withcystic fibrosis is a principal cause of their high mortality. Whenestablished, the chronic pulmonary infection is very difficult, if notimpossible, to eradicate. More than 60% of cystic fibrosis patients arecolonized with Pseudomonas aeruginosa bacterium strains which arelargely resistant to regular and specialty antibiotics, such aspiperacillin, ticarcillin, meropenem, netilmicin and only littlesensitive to azlocillin, ciprofloxacin, timentin and ceftazidime. Manystrains have also been shown to develop resistance to tobramycin and tocolistin, if used continuously.

Often, after prolonged antibiotic therapy, a superinfection withorganisms intrinsically resistant to oral, intravenous or inhaledantibiotics develops in patients with cystic fibrosis and other chronicpulmonary infections. The four most common drug resistant organisms areBurkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenesxylosoxidans, and multidrug resistant Pseudomonas aeruginosa.

Cystic fibrosis patients infected with Burkholderia cepacia have anincreased rate of mortality compared to those patients with Pseudomonasaeruginosa infections. In some cystic fibrosis patients, Burkholderiacepacia can cause a rapid fatality, as described, for example in Am. J.Respir. Crit. Care Med., 160: 5, 1572-7 (1999).

The high level of antibiotic resistance demonstrated by most strains ofBurkholderia cepacia severely limits therapeutic options for itstreatment (Clinics Chest Med., 19:473-86 (September 1998)). Furthermore,unlike Pseudomonas aeruginosa, Burkholderia cepacia can cause epidemicspread among cystic fibrosis patients and therefore any patient infectedwith Burkholderia cepacia is usually isolated from other patients. Thiscauses both additional expenses connected with caring for these patientsand may also be psychologically devastating to the patient. Furthermore,most lung transplant centers will not perform a lung transplant onpatients infected with Burkholderia cepacia (Clinics Chest Med.,19:473-86 (September 1998)). Therefore, the Burkholderia cepaciainfection is often viewed as a death sentence by patients with cysticfibrosis.

Burkholderia cepacia is usually resistant to the parenteral delivery ofvarious antibiotics, including aztreonam, with showing only 5% ofisolates to be sensitive to such treatment (Antimicrob. AgentsChemother., 34: 3, 487-8 (March 1990)). Thus it would be advantageous tohave available treatment for Burkholderia cepacia infections.

Other gram-negative bacteria intrinsically resistant to tobramycin canalso complicate the care of a cystic fibrosis patient. These bacteriainclude Stenotrophomonas maltophilia and Alcaligenes xylosoxidans.Antibiotic therapy of these infections is usually also ineffective orleads to rapid emergence of drug resistance. Therefore, the successfultreatment of all these infections requires that samples of theseisolates are sent to a laboratory for complex antibiotic synergydetermination of proper therapy for each individual patient (Ped.Pulmon., S17: 118-119 (1998)). It would, therefore, be also advantageousto provide a therapy for these rare but hard to treat bacterialinfections.

Similarly, the development of P. aeruginosa infection with strains whichare resistant to, that is which have a high minimal inhibitoryconcentration (MIC) to a majority of antibiotics including tobramycin,predicts declining lung function and also may disqualify the patientfrom consideration for lung transplant (Clinics Chest Med., 19:535-554(September 1998)).

Existing antibiotic treatments for Burkholderia cepacia,Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and multidrugresistant Pseudomonas aeruginosa pulmonary infections are eitherineffective, or lead to rapid emergence of drug resistance.

From the brief description above, it is clear that there is a continuousneed for an effective therapy for treatment of acute and chronicpulmonary bacterial infections caused by gram-negative bacteria andparticularly those caused by Burkholderia cepacia, Stenotrophomonasmaltophilia, Alcaligenes xylosoxidans, and multidrug resistantPseudomonas aeruginosa lung infections. Such therapy would preferablycomprise an inhalation of the aerosolized drug formulation delivering atherapeutically effective amount of the drug directly to theendobronchial space of airways to avoid systemic treatment. The problemsconnected with infections caused with these antibiotic resistantbacteria are very serious and it would be advantageous to have availablemore efficient modes of treatments with different types of antibiotics.

Aztreonam is a synthetic antibiotic which has a good biological activityagainst gram-negative bacteria and it has previously been used forintravenous treatment of bacterial infections. However, its use isseverely limited due to its low efficacy requiring administration ofvery large intravenous doses between 1000 and 4000 mg a day in order totreat the infections caused by gram-negative bacteria. Although it wouldbe an antibiotic of choice for complementary treatment of patientstreated with tobramycin or other antibiotics, particularly in cysticfibrosis patients, such treatment is not practical because of the highdoses required.

Moreover, aztreonam is currently only available as an arginine salt.Arginine has been shown to be toxic to the lung and causes lung tissueirritation, inflammation, bronchospasm and cough and therefore is notsuitable for a delivery by aerosolization. Consequently, aztreonamarginine salt is not approved for inhalation use in the United States orelsewhere.

However, as the antibiotic for treatment of pulmonary bacterialinfections caused by gram negative bacteria, aztreonam could become adrug of choice for such treatment, if it could be delivered byinhalation in therapeutically effective concentrations directly to thelungs and if the problems connected with the aztreonam arginine can beovercome.

However, the efficacious administration of aztreonam by inhalation isfurther complicated by a lack of safe, physiologically acceptable andstable formulations for use by inhalation. Such formulation must meetseveral criteria, such as certain size range of inhalable particles,certain pH range and certain degree of salinity. When the aerosolcontains a large number of particles with a mass medium average diameter(MMAD) larger than 5μ, these are deposited in the upper airwaysdecreasing the amount of antibiotic delivered to the site of infectionin the endobronchial space of airways. Similarly, both highly acidic andalkaline or hypotonic or hypertonic conditions lead to respiratorycomplications, such as bronchospasm and cough, preventing inhalation ofthe drug.

Thus it would be advantageous and desirable to provide an inhalableformulation for delivery of aztreonam by aerosol or a dry powderformulation for treatment of pulmonary gram-negative bacterialinfections and particularly those caused by drug resistant strainsBurkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenesxylosoxidans, and multidrug resistant Pseudomonas aeruginosa, whereinthe formulation comprises a smallest possible therapeutically effectiveamount of drug in a form which does not cause pulmonary inflammation,wherein the pH is adjusted to physiologically acceptable levels, whereinthe aqueous solution is isotonic and wherein said formulation hasadequate shelf life suitable for commercial distribution, storage anduse.

It is, therefore, a primary object of this invention to provide a methodfor treatment of gram-negative infections, especially those caused byBurkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenesxylosoxidans, and multidrug resistant Pseudomonas aeruginosa byproviding a safe, physiologically acceptable and efficacious formulationfor inhalation using a pure concentrated aztreonam free base, or apharmaceutically acceptable salt thereof, which formulation containssufficient but not excessive concentration of the active drug, whichformulation can be efficiently aerosolized by nebulization using jet,ultrasonic or atomization nebulizers, into an aerosol having particlesizes within a range from 1 to 5μ, or administered as a dry powder, bothwell tolerated by cystic fibrosis patients and by patients with impairedpulmonary function due to infections, inflammation or another underlyingdisease.

All patents, patent applications and publications cited herein arehereby incorporated by reference.

SUMMARY

One aspect of this invention is a method for treatment of pulmonaryinfections caused by gram-negative bacteria.

Another aspect of this invention is a method for treatment of pulmonarybacterial infections caused by gram-negative bacteria, said methodcomprising administration of an inhalable concentrated pure aztreonam,or a pharmaceutically acceptable salt thereof, in a dry powder form oras an aerosol containing from about 1 to about 750 mg of aztreonam, or apharmaceutically acceptable salt thereof, said aztreonam administered inan inhalable dry powder form or dissolved in from about 1 to about 5 mlof an aerosolable solution of pH between 4.5 and 7.5 containing fromabout 0.1 to about 0.9% of chloride or other anion to the lungendobronchial space of airways of a patient in need thereof bynebulization in an aerosol having a mass medium average diameter betweenabout 1 and about 5μ.

Still another aspect of this invention is a method for treatment ofpulmonary bacterial infections caused by gram-negative bacteriacomprising administering a formulation of about 1 to about 250 mg ofaztreonam once, twice, three times or four times a day up to a dailydose of aztreonam of 750 mg a day.

Yet another aspect of this invention is a method for treatment ofpulmonary bacterial infections caused by Escherichia coli,Enterobacteria species, Klebsiella pneumoniae, K. oxytoca, Proteusmirabilis, Pseudomonas aeruginosa, Serratia marcescens, Haemophilusinfluenzae, Burkholderia cepacia, Stenotrophomonas maltophilia,Alcaligenes xylosoxidans, and multidrug resistant Pseudomonas aeruginosausing an inhalable formulation of aztreonam or a pharmaceuticallyacceptable salt thereof delivered by inhalation to the endobronchialspace of airways in a dry powder form or in an aerosol.

Another aspect of this invention is an inhalable pharmaceuticallyacceptable composition comprising from about 1 to about 250 mg per onedose of aztreonam or a pharmaceutically acceptable salt thereof, saidcomposition suitable for treatment of pulmonary bacterial infectionscaused by gram-negative bacteria wherein said aztreonam or thepharmaceutically acceptable salt thereof are prepared as an inhalabledry powder or as an aerosolable solution.

Still another aspect of this invention is an aerosolized aztreonamformulation comprising from about 1 to about 50 mg/ml of aztreonam or apharmaceutically acceptable salt thereof dissolved in from about 1 to 5ml of a normal or diluted saline or another aqueous solution, having pHbetween 4.5 and 7.5.

Still another aspect of the current invention is a formulationcomprising from about 1 to about 250 mg of aztreonam in a diluted salinesolution ranging from one tenth to a half normal saline or other aqueoussolvent containing chloride or another anion, wherein said formulationhas a pH between 5.5 and 7.0 and is delivered by aerosolization in about1-5 ml of solution wherein aerosol has particles of the mass mediumaverage diameter predominantly between 1 and 5μ, wherein saidformulation is nebulized using a jet, atomizing, electronic orultrasonic nebulizer.

Still yet another aspect of the current invention is a dry powderformulation comprising from about 1 to 200 mg of aztreonam or apharmaceutically acceptable salt thereof, wherein said formulation ismilled, spray dried or precipitated into a fine powder having particleswith the mass medium average diameter between 1 and 5μ used forinhalation of the dry powder administered from one to four times per daynot exceeding 750 mg per day.

Another aspect of this invention is a two-part reconstitution systemcomprising an aztreonam in dry or lyophilized powder form and a diluentstored separately until use.

DEFINITIONS

As used herein:

“MMAD” means mass medium average diameter.

“Normal saline” means water solution containing 0.9% (w/v) NaCl.

“Diluted saline” means normal saline containing 0.9% (w/v) NaCl dilutedinto its lesser strength from about 0.1% to about 0.8%.

“Half normal saline” or “½ NS” means normal saline diluted to its halfstrength containing 0.45% (w/v) NaCl.

“Quarter normal saline” or “¼ NS” means normal saline diluted to itsquarter strength containing 0.225% (w/v) NaCl.

“One tenth normal saline” or “ 1/10 NS” means normal saline diluted toits one tenth strength containing 0.09% (w/v) NaCl.

“CF” means cystic fibrosis.

“Predominantly” means including at least 70% but preferably 90% ofparticle sizes between 1 and 5μ.

“Physiologically acceptable solution” means a saline diluted to between1/10 NS or 1 NS or another aqueous solution comprising from about 31 toabout 154 mM of chloride or an equivalent concentration of bromine oriodine.

“Composition” means an aztreonam containing formulation additionallycontaining other components, such as excipients, diluents, isotonicsolutions, buffers, etc.

“Formulation” means a specific composition formulated for specific use,such as for aerosolization of aztreonam containing solution ornebulization of dry powder.

“Aztreonam composition” or “aztreonam formulation” means a compositionor formulation comprising an indicated amount of aztreonam free base orthe equivalent of that amount in an aztreonam salt. Thus, if forexample, the dose of aztreonam comprises molar amount of aztreonam freebase, the aztreonam salt comprises equal molar amount of salt.

“Concentrated aztreonam” means an aztreonam concentrated into a formwhich permits dilution of more than 83.3 mg of aztreonam free base in 1ml of diluent.

DETAILED DESCRIPTION OF THE INVENTION

The current invention concerns a discovery that specifically formulatedand delivered inhalable aztreonam or a pharmaceutically acceptable saltthereof is efficacious for treatment of pulmonary infections caused bygram-negative bacteria.

Consequently, the invention concerns an inhalable composition and amethod of treatment for pulmonary bacterial infections caused byEscherichia coli, Enterobacter species, Klebsiella pneumoniae,Klebsiella oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, Serratiamarcescens, Haemophilus influenzae, including ampicillin-resistant andother penicillinases-producing strains and Nitrobacter species as wellas for treatment of more rare bacteria, such as Burkholderia cepacia,Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and multidrugresistant Pseudomonas aeruginosa. The aztreonam formulation or aformulation comprising a pharmaceutically acceptable salt thereof isdelivered to a patient's endobronchial space of airways by inhalation ofa dry powder or an aerosol solution.

The method of treatment of pulmonary bacterial infections is especiallysuitable for treatment of patients with cystic fibrosis, bronchiectasisand patients with pneumonia assisted by ventilators, however it is alsouseful for treatment of other conditions that are complicated byinfections caused by Burkholderia cepacia, Stenotrophomonas maltophilia,Alcaligenes xylosoxidans, and multidrug resistant Pseudomonas aeruginosaor other gram-negative bacteria.

The current invention thus concerns a novel, efficacious, safe,nonirritating and physiologically compatible inhalable aztreonamcomposition suitable for treatment of pulmonary bacterial infectionscaused by gram-negative bacteria particularly those which are resistantto treatment with other antibiotics. The inhalable formulation ofaztreonam or a pharmaceutically acceptable salt thereof is suitable bothfor treatment and prophylaxis of acute and chronic pulmonary infections.The inhalable formulation is delivered as an aerosol or as an inhalabledry powder. For aerosolization, aztreonam is dissolved in a minimalvolume of about 1 to about 5 ml of an aqueous solvent comprisingchloride bromine or iodine ion, having a pH between 4.5 and 7.5,delivered to the endobronchial space in an aerosol having mass mediumaverage diameter particles predominantly between 1 to 5μ using anebulizer able to aerosolize the aztreonam solution into particles ofrequired sizes.

I. Aztreonam and Pharmaceutically Acceptable Salts Thereof

Aztreonam is a known synthetic monocyclic monobactam antibiotic withantibacterial activity against most gram-negative bacteria. Aztreonamarginine salt, known under its trade name AZACTAM® is currently FDAapproved only for intravenous and intramuscular use.A. Aztreonam Compound

Aztreonam chemical formula is(Z)-2-[[[(2-amino-4-thiazolyl)[[(2S,3S)-2-methyl-4-oxo-1-sulfo-3-azetidinyl]carbamoyl]methylene]amino]oxy]-2-methylpropionicacid.

Aztreonam is a monobactam and as such it has a unique monocyclicbeta-lactam nucleus, and is therefore structurally different from otherβ-lactam antibiotics such as, for example penicillins, cephalosporins,or cephamycins.

The sulfonic acid substituent in the 1-position of the ring activatesthe beta-lactam moiety. An aminothiazolyl oxime side chain in the3-position and a methyl group in the 4-position confer the specificantibacterial spectrum and beta-lactamase stability.

AZACTAM® (aztreonam for injection, USP) commercially available from DURAPharmaceuticals, Inc., San Diego, Calif., contains aztreonam as theactive ingredient. AZACTAM® is supplied as a sterile, nonpyrogenic,sodium-free, white to yellowish-white lyophilized powder containingarginine. AZACTAM is formulated for intramuscular or intravenous use.(PDR, pg. 1159 (2001)).

The commercially available AZACTAM intravenous or intramuscularformulation is not suitable for inhalable use because of the presence ofarginine in the formulation. Arginine has been found to cause pulmonaryinflammation when administered in aerosol form to the lung in the rat.

Arginine has been unsuccessfully used as a potential aerosolizedmucolytic agent in cystic fibrosis patients. A study, described inPediatrics, 55:96-100 (1975) recommends that arginine should not be usedin cystic fibrosis patients. In a study of 24 patients with cysticfibrosis, inhalation therapy with an arginine solution in five patientshad to be stopped because of severe deterioration of their generalconditions and the appearance of cough. The presence of airwayinflammation in these five patients was confirmed by bronchoscopy. Sincethan, it has been discovered that arginine is a substrate for theproduction of nitric oxide radicals.

Nitric oxide radical reacts with the superoxide anion to formperonitrile, which is by itself toxic to the tissue and also may furtherreact to form highly reactive and toxic hydroxyl radical. Sinceinflammation is a serious impairment for cystic fibrosis and all otherdiseases which this invention attempts to treat, use of arginine saltwould defeat this purpose as it would worsen rather than better thepatient conditions.

Arginine is also an important substrate for immune complex injury in thelung, as disclosed in PNAS, 14:6338-6342 (1991). Since theaerosolization concentrates high levels of the aerosolized drug in thelung as compared to dilution seen after intravenous administration, theaerosolization of the aztreonam arginine salt would be detrimentalrather than advantageous treatment for cystic fibrosis patients orpatients suffering from pulmonary infections. Moreover, it would diluteand/or negate the effect of aztreonam.

Aztreonam is not currently approved or used for inhalation treatment andaerosol administration in the United States. Consequently, there is noknown aztreonam containing formulation available for aerosol delivery ofaztreonam to the endobronchial space of airways. The only attempt todeliver aztreonam intermittently to cystic fibrosis subjects isdescribed in Spanish Annals on Pediatrics, 40: No.3 (1994) where suchdelivery was made in an open label trial in cystic fibrosis patientswith intermittently administered 500 and 1000 mg of AZACTAM USP argininesalt, twice a day for 21 days, using CR60 System 22 unit nebulizer. Theintent of this study was to treat aztreonam sensitive Pseudomonasaeruginosa organisms, but not multidrug resistant Pseudomonasaeruginosa. No effort or speculation was to treat Burkholderia cepacia,Stenotrophomonas maltophilia, infections caused by Alcaligenesxylosoxidans or other gram-negative bacteria.

In this study, the nebulized solution of aztreonam was delivered afterthe physical therapy session. Prior to the therapy session, the patientswere administered 3 cc of saline alone or mixed with bronchodilatorssalbutamol or ipratropium bromide and fenoterol bromohidrate to preventbronchospasm. The treatment described in this study thus required boththe pretreatment with inhaled saline and/or bronchodilating agents andprior physical therapy session as well as administration of large dosesof the drug to be administered twice a day. Although in about 80% ofpatients lung function has somehow improved, such improvement was notstatistically significant. At least one patient could not tolerate thetherapy due to bronchospasm. Most patients required administration ofbronchodilators and all patients underwent physical therapy prior toaztreonam treatment in order to tolerate the administration of largedoses of nebulized aztreonam. Aztreonam therapy was discontinued if invitro resistance was found. One patient developed Burkholderia cepacia,which was viewed as superinfection, and a possible adverse outcome. Thereference, although suggestive of efficacy in drug sensitive Pseudomonasaeruginosa, which is expected because the drug is known for its effecton the gram-negative bacteria, does not disclose the use of low doses ofaztreonam, or its continuous use, or the use of aztreonam salts which donot cause bronchospasm, or the use of aztreonam for treatment ofmultidrug resistant P. aeruginosa and teaches away from use inBurkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenesxylosoxidans, and multidrug resistant Pseudomonas aeruginosa.Furthermore, the high incidence of bronchospasm developed with use ofthe disclosed formula requiring either discontinuation or pretreatmentwith bronchodilators indicates the need for a different formulation safefor inhalation use.

B. Aztreonam Biological and Pharmacological Activity

Aztreonam exhibits potent and specific activity in vitro against a widespectrum of gram-negative aerobic pathogens including Pseudomonasaeruginosa. The bactericidal action of aztreonam results from theinhibition of bacterial cell wall synthesis due to a high affinity ofaztreonam for penicillin binding protein 3 (PBP3).

Aztreonam, unlike the majority of β-lactam antibiotics, does not induceβ-lactamase activity and its molecular structure confers a high degreeof resistance to hydrolysis by β-lactamases, such as penicillinases andcephalosporinases, produced by most gram-negative and gram-positivepathogens. Aztreonam is therefore especially active againstgram-negative aerobic organisms that are resistant to antibioticshydrolyzed by β-lactamases.

Aztreonam maintains its antimicrobial activity at a pH ranging from 6 to8 in vitro (AZACTAM® product label, Dura Pharmaceuticals), as well as inthe presence of human serum and under anaerobic conditions. Aztreonam isactive in vitro and is effective in laboratory animal models andclinical infections against most strains of the following organisms,Escherichia coli, Enterobacter species, Klebsiella pneumoniae,Klebsiella oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, Serratiamarcescens, Haemophilus influenzae, and Nitrobacter species, includingmany that are multi-resistant to other antibiotics such as certaincephalosporins, penicillins, and aminoglycosides.

Aztreonam is thus suitable for treatment of infections caused byEscherichia coli, Enterobacter species, Klebsiella pneumoniae,Klebsiella oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, Serratiamarcescens, Haemophilus influenzae, Nitrobacter species.

Currently, the only infections for which AZACTAM is FDA approved arethose caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonasaeruginosa, Haemophilus influenzae, Proteus mirabilis, Enterobacterspecies and Serratia marcescens.

It has now been found that all the above named bacterial strains as wellas rare and very resistant strains, such as Burkholderia cepacia,Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and multidrugresistant Pseudomonas aeruginosa are successfully eradicated by dailytreatment with low doses between about 1 and about 250 mg of aztreonamfree base or a pharmaceutically acceptable salt thereof, preferablyadministered once or twice a day, with total daily doses not exceeding750 mg/day.

II. Aztreonam Pharmacologically Acceptable Salts

Currently, the only commercially available salt of aztreonam isarginine. As already discussed above, the aztreonam salt is not suitablefor inhalation administration because arginine, after aerosol exposure,is known to cause pulmonary inflammation, bronchospasm and cough.AZACTAM, aztreonam containing arginine salt, is not approved byregulatory authorities for inhalation use. Therefore, other aztreonamsalts are needed to achieve a safe formulation of aztreonam forinhalation treatment of patients with pulmonary infections or thosehaving impaired pulmonary function due to cystic fibrosis orbronchiectasis.

Since the aztreonam containing arginine is not suitable for inhalationaccording to this invention, other acid addition salts were preparedaccording to Example 3, tested and found pharmacologically acceptableand without detrimental secondary effects when administered as a drypowder or aerosol.

The aztreonam for use in the current invention is prepared in the formof salts derived from inorganic or organic acids. These salts includebut are not limited to the following salts: acetate, adipate, alginate,citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,camphorate, camphorsulfonate, digluconate, cyclopentanepropionate,dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, nicotinate, 2-napthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate andundecanoate.

Examples of acids which may be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulphuric acid and phosphoric acid and such organicacids as, for example, oxalic acid, maleic acid, acetic, aspartic,succinic acid and citric acid. Basic addition salts can be prepared insitu during the final isolation and purification of the compound offormula (I), or separately by reacting the carboxylic or sulfuric acidfunction with a suitable base such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation or withammonia, or with an organic primary, secondary or tertiary amine.

Pharmaceutical acceptable salts also include, but are not limited to,cations based on the alkali and alkaline earth metals, such as sodium,lithium, potassium, calcium, magnesium or aluminum salts and the like,as well as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, amino acids including basic amino acids (i.e.lysine, histidine, ornithine) and the like. Other representative organicamines useful for the formation of base addition salts includediethylamine, ethylenediamine, ethanolamine, diethylamine and the like.

Any of the above named salt may be delivered as a single salt or asadmixture of one or several salts as long as the equivalent amount ofaztreonam is within 1 to 250 mg per one dosage.

The preferred pharmaceutically acceptable aztreonam salt is derived fromreaction of aztreonam with hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid and phosphoric acid as these salts are not known tocause pulmonary inflammation and are safer than arginine salts.

III. Aztreonam Inhalable Composition

The current invention primarily concerns a concentrated inhalableaztreonam composition suitable for efficacious delivery of aztreonam orthe aztreonam pharmaceutically acceptable salt into the endobronchialspace of airways by aerosolization or as a dry powder.

The invention is most preferably suitable for formulation ofconcentrated aztreonam for aerosolization by atomizing, jet, ultrasonic,pressurized, vibrating porous plate or equivalent nebulizers or by drypowder inhalers which predominantly produce aztreonam aerosol or drypowder particles between 1 and 5μ. Such particle sizes are necessary forefficacious delivery of aztreonam into the endobronchial space to treatbacterial infections.

A. Aerosolized Aztreonam Composition

Aztreonam composition for aerosolization is formulated for efficaciousdelivery of aerosolized aztreonam to the lung endobronchial space ofairways.

The aerosol formulation is delivered in a total volume of between about1 and about 5 ml of aqueous physiologically acceptable solution for oneinhalation dose. When formulated and delivered according to the methodof invention, it delivers a therapeutically efficacious dose ofaztreonam to the site of the infection in amount of aztreonam sufficientto treat bacterial pulmonary infections.

A combination of the novel aqueous formulation with the atomizing, jet,pressurized, vibrating porous plate or ultrasonic nebulizer permits,depending on the nebulizer, about at least 20 to about 90%, typicallyabout 70% delivery of the administered dose of aztreonam into airways.

The formulation contains a minimal yet efficacious amount of aztreonamfrom 1 to about 250 mg formulated in the smallest possible volume ofphysiologically acceptable diluent having a certain degree of salinityand certain pH, adjusted to permit generation of an aztreonam aerosolwell tolerated by patients but minimizing the development of secondaryundesirable side effects such as bronchospasm and cough.

Primary requirements for aerosolized aztreonam formulation are itssafety and efficacy. Additional advantages are lower cost, practicalityof use, long shelf-life, storage and manipulation of the aerosol device.These requirements for aerosolized aztreonam have now been found to bemet by the formulation containing certain degree of salinity and havecertain pH range.

a. Dosage of Aztreonam or Salt Thereof

Aztreonam has relatively short life-time. Consequently, the effectivetreatment of bacterial pulmonary infections requires a treatment regimenwhich provides sufficient amount of drug to maintain the antibacteriallevel of aztreonam in the lung. Such regimen thus requiresadministration of an inhalable aztreonam one to several, preferably twoto four, times a day. Most preferred dosing regimen for patientconvenience is once or twice a day, however, because of a specificeffect aztreonam asserts on the bacteria, and because of its relativelyshort life-time of about 12 hours, more than twice a day dosing is oftenrequired for complete eradication of the bacteria from the endobronchialspace.

It is therefore preferable to deliver aerosolized or dry powderaztreonam or a pharmaceutically acceptable salt thereof in the smallesttherapeutically efficacious amount at least twice a day, in someinstances three to four times, and exceptionally more than four times aday. A dose of aztreonam or a salt thereof is therefor set to be between1 and 250 mg per one dose. Typically, one therapeutically effective dosecontains between 1 and 250 mg of aztreonam free base or the saltthereof, in equivalent. Typically, the formulation and the nebulizer areselected to provide at least about 50%-70% efficacy of aztreonamdelivery to the endobronchial space. noninvasive administration of smallyet effective amounts of aztreonam directly into lungs is greatimprovement compared to all previously known method used for delivery ofaztreonam.

2. Effect of pH on Aztreonam Aerosol Formulation

The solution or diluent used for preparation of aztreonam aerosol has alimited pH range from 4.5 to 7.5, preferably between 5.5 and 7.0.

The pH of the formulation is an important feature for aerosolizedaztreonam delivery. When the aerosol is either acidic or basic, it cancause bronchospasm and cough. Although the safe range of pH is relativeand some patients may tolerate a mildly acidic aerosol, others,particularly those with,cystic fibrosis or other underlying disease willexperience bronchospasm. Any aerosol with a pH of less than 4.5typically induces bronchospasm. Aerosols with a pH between 4.5 and 5.5will cause bronchospasm occasionally. Testing with aztreonam aerosoldiscovered that an aerosolizable aztreonam formulation having a pHbetween 5.5 and 7.0 is well tolerated and safe. Any aerosol having pHgreater than 7.5 is to be avoided as the body tissues are unable tobuffer alkaline aerosols. Aerosol with controlled pH below 4.5 and over7.5 result in lung irritation accompanied by severe bronchospasm coughand inflammatory reactions.

For these reasons as well as for the avoidance of bronchospasm, cough orinflammation in patients, the optimum pH for the aerosol formulation wasdetermined to be between pH 5.5 to pH 7.0.

Consequently the aztreonam aerosol formulation is adjusted to pH between4.5 and 7.5 with preferred pH range from about 5.5 to 7.0. Mostpreferred pH range is from 5.5 to 6.5.

3. Effect of Salinity on the Aztreonam Formulation

Patients suffering from acute or chronic endobronchial infections andparticularly those with cystic fibrosis or bronchiectasis have increasedsensitivity to various chemical agents and have high incidence ofbronchospastic, asthmatic or cough incidents. Their airways areparticularly sensitive to hypotonic or hypertonic and acidic or alkalineconditions and to the presence of any permanent ion, such as chloride.Any imbalance in these conditions or a presence of chloride abovecertain value leads to bronchospastic or inflammatory events and/orcough which greatly impair treatment with inhalable formulations. Boththese conditions prevent efficient delivery of aerosolized aztreonaminto the endobronchial space. The clinical manifestations of theirritated airways are extremely undesirable.

Clearly, for aztreonam, it is not possible to use solely an aqueoussolvent without providing certain degree of osmolality to meet andemulate physiological conditions found in healthy lungs. Consequently,certain amount of the chloride or another anion is needed for successfuland efficacious delivery of aerosolized aztreonam but such amount ismuch lower than amounts provided and typically used for aerosols ofother compounds.

Bronchospasm or cough reflexes do not respond to the same osmolality ofthe diluent for aerosolization, however, they can be sufficientlycontrolled and/or suppressed when the osmolality of the diluent is in acertain range. Preferred solution for nebulization of aztreonam which issafe and has airways tolerance has a total osmolality between 50 and 550mOsm/kg with a range of chloride concentration of between 31 mM and 300mM. The given osmolality controls bronchospasm, the chlorideconcentration, as a permeant anion, controls cough. In this regard thechloride anion can be substituted with bromine or iodine anions, sinceboth are permeant anions. In addition, bicarbonate may be wholly orpartially substituted for chloride ion. Normal saline (NS) contains 154mM of chloride whereas 31 mM of chloride corresponds to about 0.2 normalsaline.

Consequently, the formulation for aztreonam aerosol of the inventioncomprises from about 1 to about 50 mg, preferably about 10 mg, ofaztreonam dissolved in 1 ml of a normal, or preferably a diluted salineto from about 1/10 normal saline (NS) to about and at most to 1 NSsolution, preferably from about 1/10 to about ¼ NS, that is a one tenthto one quarter diluted normal saline. It has now been discovered thataztreonam is efficaciously delivered into lungs when dissolved in lesserthan normal saline, that is saline containing 0.9% of sodium chloride,and that the concentration of a chloride ion equal to or lesser than ¼ Nsaline permits and assures a delivery of aztreonam into endobronchialspace.

The aztreonam formulation containing about 50 mg of aztreonam per 1 mlof 0.2 NS has an osmolality of about 290 mOsm/l. Such osmolality iswithin a safe range of aerosols suitable for administration to patientssuffering from pulmonary bacterial infections and also those patientswith a cystic fibrosis or bronchiectasis.

An additional feature and advantage of using 1/10 to ¼ NS solutioncomprising 50 mg/ml aztreonam is that the resulting aerosol formulationis very efficiently nebulized by an atomic, jet or ultrasonic nebulizercompared to aztreonam dissolved in a normal saline. Since the deliveryof aztreonam formulated as described herein is much more efficient, muchlower amount of aztreonam is needed to achieve complete eradication ofgram-negative bacteria in lungs. Instead of 1000 to 4000 mg of aztreonamwhich was shown to be somehow effective in the only one prior attempt toaerosolize aztreonam, the formulation of aztreonam according to thisinvention permits treatments with as little as 1 mg/ml and with at mostup to 50 mg/ml of aztreonam in a maximum amount of 5 ml volume,delivered preferably with an atomizing, jet, electronic or ultrasonicnebulizer.

4. Preferred Aerosolizable Aztreonam Formulation

The aztreonam aerosolizable formulation comprises aztreonam or apharmaceutically acceptable salt thereof in amount about 1 to about 50mg/ml of about 1 to 5 ml of an aqueous solution containing lowconcentration of chloride ion, having pH adjusted to between 4.5 and7.5, said formulation delivered by aerosolization using an atomizing,jet, electronic, ultrasonic nebulizer.

The preferred formulation of the current invention is a formulationcomprising from about 10 to about 50 mg of aztreonam dissolved in about1-5 ml of a saline diluted preferably to a quarter or one tenth strengthof normal saline, having pH adjusted to between 5.5 and 7.0, deliveredby nebulization in aerosol particles having the mass medium averagediameter predominantly between 1 and 5μ, wherein said formulation isnebulized using an atomizing, jet, electronic or ultrasonic nebulizer.

The formulation according to the invention contains aztreonam formulatedas a dry powder, aerosol solution or aerosol suspension of liposomes orother microscopic particles in an aqueous solvent. The formulation isdesigned to be well tolerated and able to be reliably and completelynebulized to aerosol particles within the respirable size range of 1 to5μ.

The doses are designed to contain as much as, but not more than, thenecessary amount of a most active form of aztreonam to preventcolonization and/or to treat severe pulmonary infections caused by arange of susceptible gram-negative organisms.

Patients can be sensitive to pH, osmolality, and ionic content of anebulized solution. Therefore these parameters are adjusted to becompatible with aztreonam chemistry and still tolerable to patients.

The formulation of the invention is nebulized predominantly intoparticle sizes allowing a delivery of the drug into the terminal andrespiratory bronchioles where the bacteria reside during infection andin the larger airways during colonization.

For efficacious delivery of aztreonam to the lung endobronchial space ofairways in an aerosol particle, the formation of an aerosol having amass medium average diameter predominantly between 1 to 5μ is necessary.The formulated and delivered amount of aztreonam for treatment andprophylaxis of endobronchial bacterial infections must effectivelytarget the lung surface. The formulation must have a smallest possibleaerosolizable volume able to deliver an effective dose of aztreonam tothe site of the infection. The formulation must additionally provideconditions which would not adversely affect the functionality of theairways. Consequently, the formulation must contain enough of the drugformulated under the conditions which allow its efficacious deliverywhile avoiding undesirable reactions. The new formulation according tothe invention meets all these requirements.

B. Aztreonam Dry Powder Composition

An alternative way to deliver inhalable aztreonam is by way of dryinhalable powder.

The aztreonam antibiotic compounds of the invention may beendobronchially administered in a dry powder formulation for efficaciousdelivery of the finely milled antibiotic into the endobronchial spaceusing dry powder or metered dose inhalers as an alternative to aerosoldelivery.

A dry powder formulation has potency, on a mass basis, which allows suchalternative delivery of aztreonam as a dry powder using dry powderinhaler. A sufficiently potent formulation of aztreonam, or apharmaceutically acceptable salt thereof, provides a dry powder whichcan be advantageously delivered by dry powder inhaler or by metered doseinhaler. For delivery of dry inhalable powder, aztreonam is milled,precipitated, spray dried or otherwise processed to particle sizesbetween about 1 and 5μ.

Dry powder formulation comprises from about 20 to 200 mg, preferably 10to 100 mg of aztreonam or a pharmaceutically acceptable salt thereof.

For dry powder formulation of the invention, aztreonam, or apharmaceutically acceptable salt thereof, is milled to a powder havingmass median average diameters ranging from 1-5 microns by media milling,jet milling, spray drying or particle precipitation techniques.

Particle size determinations are made using a multi-stage Andersoncascade impactor or other suitable method. The Thermo Andersen EightStage Non-Viable Cascade Impactor is specifically cited within the USPharmacopoeia Chapter 601 as a characterizing device for aerosols withinmetered-dose and dry powder inhalers. The Eight Stage Cascade Impactorutilizes eight jet stages enabling classification of aerosols from 9.0micrometers to 0.4 micrometers (at 28.3 L/min) and allows airborneparticulate to impact upon stainless steel impaction surfaces or avariety of filtration media substrates. A final filter collects allparticles smaller than 0.4.

Media milling is accomplished by placing a drug substance into a millcontaining, for example, stainless steel or ceramic balls and rotatingor tumbling the material until the desired drug particle size ranges areachieved. Advantages of media milling include good size control, narrowproduct size ranges, high efficiencies of recovery, and readily scalableprocesses. Disadvantages include long manufacturing process times whichtakes from several hours to several days, the requirement that themilling media be separated from the product at completion, and thepossibility of contamination of the product with the media.

Jet milling uses very high pressure air streams to collide particleswith one another, with fine particles of the desired size beingrecovered from the mill. Advantages include rapidity of themanufacturing process and less energy transfer during milling, resultingin less temperature rise during the drug production. The jet millingprocess is completed in seconds to minutes. Disadvantages of the jetmilling include poorer yield and collection efficiencies, with only 50to 80% of recovery being a typical yield.

Spray-drying is another technique useful for preparation of inhalabledry powder. Spray drying involves spraying a fine mist of aztreonamsolution onto a support and drying the particles. The particles are thencollected. Spray drying has the advantage of being the least prone todegrading chemical entities. Adding a co-solvent which decreases thesolubility of a drug to a uniform drug solution results in solutionprecipitation. When sufficient co-solvent is added, the solubility ofthe drug falls to the point where solid drug particles are formed whichcan be collected by filtration or centrifugation. Precipitation has theadvantage of being highly reproducible, having a high yield of recoveryand being able to be performed under low temperature conditions, whichreduce degradation.

Dry powder inhalation and metered dose inhalations are more practicalwhen administered doses result in the delivery of at least about 10 mg,and more preferably about 25 to about 100 mg, of aztreonam antibioticcompound to the lung of the patient receiving treatment. Depending onthe efficiency of the dry powder delivery device, which is typicallyabout 70%, typical effective dry powder dosage levels fall in the rangeof about 20 to about 60 mg of aztreonam. Therefore, typically more thanone breath of drug is required.

In this aspect, the invention provides a sufficiently potent formulationof pure aztreonam antibiotic or a pharmaceutically acceptable salt indry powder or metered dose form of drug particles milled or otherwiseprepared to particle sizes predominantly with a range of 1 to 5 microns.Such formulation is practical and convenient because it does not requireany further handling such as diluting the dry powder or filling anaerosol container. Further, it utilizes the devices that aresufficiently small, fully portable and do not require, for example, anair compressor which is needed for a jet nebulizer. Additionally, thedry powder formulation has a longer shelf life that the liquid aztreonamformulations for aerosolization. Aztreonam, when reconstituted into anaerosolizable solution, has only a limited shelf life at roomtemperature due to hydrolysis of the monobactam ring. Aztreonam drypowder does not have this problem.

The dry powder formulation is thus practical and convenient forambulatory use because it does not require dilution or other handling,it has an extended shelf-life and storage stability and the dry powderinhalation delivery devices are portable and do not require an aircompressor needed by aerosol nebulizers.

All techniques suitable for preparation of dry inhalable powders and anyand all improvements thereof as well as any dry powder inhaler areintended to be within the scope of the invention.

C. Shelf-Life and Storage

Stability of the formulation is another very important issue forefficacious formulation. If the drug is degraded before aerosolization,a smaller amount of the drug is delivered to the lung thus impairing thetreatment efficacy. Moreover, degradation of stored aztreonam maygenerate materials that are poorly tolerated by patients.

The dry form of aztreonam has at least 2 years long shelf life. Theliquid forms of the arginine/aztreonam free base have a 24-hourstability at room temperature, 48 hours when refrigerated, and whenfrozen at −4° C., such stability can be extended to about three months.However, the stability of aztreonam arginine salt is an attribute ofarginine. The stability of other salts, after liquid reconstitution maydiffer.

A long-term stability of aztreonam free base or aztreonam salt inaqueous solutions may not provide a sufficiently long shelf life whichwould be commercially acceptable. A liquid formulation, therefore, mayrequire a separation of aztreonam or aztreonam salt from the appropriatediluent. For this reason, the formulation is preferably supplied in adry form and can be a reconstituted prior to administration.

A formulation for aerosolization is thus preferably provided as twoseparate components, one containing a dry aztreonam or a salt thereofand a second containing an appropriate diluent such as 0.1 to 0.9 Nsaline, bicarbonate or any equivalent acqueous solution, as describedabove. The formulation is reconstituted immediately prior toadministration. This arrangement prevents problems connected with thelong-term stability of aztreonam in aqueous solvents.

According to the invention, aztreonam for aerosolization is preferablyformulated in a lyophilized dosage form intended for use as a dry powderfor reconstitution before inhalation therapy. The formulation ofaztreonam can be aseptically prepared as a lyophilized powder either fordry powder delivery or for reconstitution and delivery, or as a frozensolution, a liposomal suspension, or as microscopic particles. Thestorage suitability of the formulation allows reliable reconstitution ofthe formulated aztreonam suitable for aerosolization.

IV. Administration of Aztreonam by Inhalation

Aztreonam is currently only available for parenteral use in the form ofthe arginine salt. Arginine is known to cause pulmonary inflammation andirritation, as discussed above, and is, therefore, unsuitable forinhalation use.

A. Two Modes of Inhalable Administration

Administration of inhalable aztreonam is achieved either with aztreonamaerosol or with inhalable dry aztreonam powder.

An arginine free formulation according to the invention delivered byinhalation, however, has been shown to safely treat respiratoryinfections caused by all susceptible gram-negative bacteria includingPseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae,Pseudomonas aeruginosa, Haemophilus influenzae, Proteus mirabilis,Enterobacter species and Serratia marcescens, as well as, and moreimportantly, antibiotics resistant strains Burkholderia cepacia,Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and multidrugresistant Pseudomonas aeruginosa.

B. Frequency of Dosing

The frequency of dosing is another aspect of this invention. Treatmentof pulmonary infections caused by the above named bacteria is achievedby a treatment regimen which provides one to several, preferably four,times a day an inhalable aztreonam. Most preferred dosing regimen forpatient covenience is once or twice a day, however, because of aspecific effect aztreonam asserts on the bacteria, and because of itsrelatively short life-time of about 12 hours, more often dosing is oftenrequired for complete eradication of the bacteria from the endobronchialspace.

In patients with severely impaired lung function, the frequency ofdosing may be increased up to about twelve times a day each time,providing only such amount of aztreonam as necessary to maintaintherapeutic level in the lung.

Aztreonam kills bacteria by lysing cell walls as long as the localconcentration of antibiotic exceeds the bacteria minimal inhibitoryconcentration (Med. Clinics N. Am., 79: 4, 733-743 (1995)). Because ofthe relatively rapid clearance of antibiotics from the respiratory tractdue to mucociliary action, greater efficacy is obtained at a lower doseof administered aztreonam by treating a patient three, four or moretimes a day rather than administer the drug only once or twice. To thiseffect the dose delivered by inhalation is at least four times and canbe one thousand time lower then the dose delivered intravenously orutilized in the one attempt described above to deliver aztreonam byaerosolization where 500-1000 mg was delivered twice a day to a totalamount of 1000 mg for children under 5 years of age and 2000 mg forindividuals older than 5 years.

The current daily dose can be as small as 2 mg. The typical upper limitis 500 mg of aztreonam per day delivered in two to four administrations.In extreme cases the dose may reach up to 750 per day delivered inthree, four or more aerosol administrations. Typical and preferred rangefor one aerosol dosage is between 20 and 200 mg administered twice a dayor between 10 and 100 mg administered three or four times per day. Fordry powder inhalation, the dose for one administration is lower,typically between about 10 and 100 mg per one dose and at maximum canreach 200 mg per one dose.

Aerosolization of aztreonam utilizes delivery of aerosolized aztreonamor a pharmaceutically acceptable salt thereof, or a mixture of saltsusing atomizing, jet, ultrasonic, electronic or other equivalentnebulizers. Those which are portable, such as atomizing, ultrasonic andelectronic nebulizers are preferred for ambulatory treatment. The jetnebulizers with a compressor nebulize the aztreonam formulation veryefficiently but are more suitable for use in the hospital and doctor'soffice.

A dry powder inhalation, as the second mode of administration of theinhalable aztreonam, also utilizes the aztreonam, or a pharmaceuticallyacceptable salt or a mixture thereof, but aztreonam is formulated as anaztreonam dry powder formulation. Such formulation comprises a deliveryof the finely milled aztreonam directly to the endobronchial space. Inthis instance, aztreonam is delivered into the endobronchial space usingdry powder or metered dose inhalers. The aztreonam potency, determinedon a mass basis, allows the inhalation of aztreonam powder, as analternative mode of administration to the aerosol. Dry powder inhalationis most efficacious, practical and economical when administered dosescontain less than 100 mg. The frequency of dosing, thus, is typicallythree or four times a day but also includes one or two or more than fourtimes dosing regimen as this regimen depends on the need and conditionof the patient.

The invention provides a sufficiently potent formulation of aztreonam ora pharmaceutically acceptable salt thereof in a form of dry powderdelivered as metered dose inhalation of aztreonam particles milled orspray dried to particle sizes predominantly within a range of 1 to 5μ.Such dry powder delivery is possible and preferable particularly forambulatory inhalation as it simplifies the delivery process. Suchdelivery is convenient because it does not require any further handlingsuch as diluting the dry powder or mixing the powder with a solvent,etc. Further, the dry powder inhalation utilizes the devices that aresufficiently small, fully portable and do not require, for example, anair compressor which is needed for a jet nebulizer. Additionally, thedry powder formulation has even longer shelf life than the liquidaztreonam formulation for aerosolization.

The dosing regimen for both aerosol and dry powder aztreonam comprisesfrom one to four, typically, or more than four times daily, in untypicalcases, administration of the aerosol or dry powder.

Severely impaired cystic fibrosis patients, for example, may be able towithstand only one inhalation at a time but could repeat this inhalationof small amount of aztreonam every two, three or four hours to obtainsufficient level of aztreonam in the lungs.

V. Devices for Delivery of Aerosolized Aztreonam

A primary requirement of this invention is to deliver aztreonamefficiently to the endobronchial space of airways in economic way. Thus,the invention requires that at least 30-50%, preferably 70-90% of theactive drug, that is aztreonam or a salt thereof, subjected tonebulization is in fact delivered to a site where it asserts itstherapeutic effect.

a. Nebulizers

The composition of the invention described above provides the drugformulated in a solution permitting delivery of a therapeuticallyefficient amount of the drug, provided that the aerosol generated by thenebulization meets criteria required for such efficient delivery. Theapparatus (nebulizer) which aerosolizes the formulation of aztreonamthus becomes a very important part of the invention.

There are quite a few nebulizer types currently commercially available.Not all of them are suitable for practicing this invention.

A nebulizer is selected primarily on the basis of allowing the formationof aztreonam aerosol having a mass medium average diameter predominantlybetween 1 to 5μ. The delivered amount of aztreonam must be efficaciousfor treatment and prophylaxis of endobronchial infections, particularlythose caused by susceptible bacteria. The selected nebulizer thus mustbe able to efficiently aerosolize the formulation which has salinity,osmotic strength, and pH adjusted as to permit generation of aztreonamaerosol that is therapeutically effective and well tolerated bypatients. The negulizer must be able to handle the formulation having asmallest possible aerosolizable volume and still able to delivereffective dose of aztreonam to the site of the infection. Additionally,the aerosolized formulation must not impair the functionality of theairways and must minimize undesirable side effects.

The inability of certain nebulizers to nebulize therapeutic quantitiesof drugs into small and uniform particle size aerosols is well known.For efficacious delivery of aztreonam a range of aerosolized particleswith MMAD needed to deliver the drug to the endobronchial space, thesite of the infection, is between 1-5μ. Many commercially availablenebulizers are able to aerosolize large volumes of the solution with anaim to deliver at least 10% of the volume to the endobronchial space byproducing around 90% of large aerosol particles above 5μ with a verylarge number of particles being in the range of 50-100μ. Thesenebulizers are inefficient and not suitable for delivery of aztreonamaccording to this invention.

In order to be therapeutically effective, the majority of aerosolizedaztreonam particles should not have larger mass medium average diameter(MMAD) than between 1 and 5μ. When the aerosol contains a large numberof particles with a MMAD larger than 5μ, these are deposited in theupper airways decreasing the amount of antibiotic delivered to the siteof infection in the lower respiratory tract.

Previously, two types of nebulizers, jet and ultrasonic, have been shownto be able to produce and deliver aerosol particles having sizes between1 and 5μ. These particle size are optimal for treatment of pulmonarybacterial infection cause by gram-negative bacteria such as Pseudomonasaeruginosa, Escherichia coli, Enterobacter species, Klebsiellapneumoniae, K. oxytoca, Proteus mirabilis, Pseudomonas aeruginosa,Serratia marcescens, Haemophilus influenzae, Burkholderia cepacia,Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and multidrugresistant Pseudomonas aeruginosa. However, unless a specially formulatedsolution is used, these nebulizers typically need larger volumes toadminister sufficient amount of drug to obtain a therapeutic effect.Therefore, without a specially formulated aztreonam the efficientdelivery of aztreonam is not achieved.

Nebulizer suitable for practicing this invention must be able tonebulize a small volume of the formulation efficiently, that is into theaerosol particle size predominantly in the range from 1-5μ.Predominantly in this application means that at least 70% but preferablymore than 90% of all generated aerosol particles are within 1-5μ range.

Jet and ultrasonic nebulizers can produce and deliver particles betweenthe 1 and 5μ particle size. A jet nebulizer utilizes air pressurebreakage of an aqueous aztreonam solution into aerosol droplets. Anultrasonic nebulizer utilizes shearing of the aqueous aztreonam solutionby a piezoelectric crystal.

Typically, however, the jet nebulizers are only about 10% efficientunder clinical conditions, while the ultrasonic nebulizer are only about5% efficient. The amount deposited and absorbed in the lungs is thus afraction of the 10% in spite of the large amounts of the drug placed inthe nebulizer.

One type of nebulizer which is suitable and preferred for aztreonamdelivery is an atomizing nebulizer which consists of a liquid storagecontainer in fluid contact with the diaphragm and inhalation andexhalation valves. For administration of the aztreonam formulation, 1 to5 ml of the formulation is placed in the storage container, aerosolgenerator is engaged which produces atomized aerosol of particle sizesselectively between 1 and 5μ.

Typical nebulizing devices suitable for practicing this inventioninclude atomizing nebulizers, or modified jet nebulizers, ultrasonicnebulizers, electronic nebulizers, vibrating porous plate nebulizers,and energized dry powder inhalers modified for handling small volume ofhighly concentrated drug in a specific formulation having a specific pH,osmolality and salinity. Most preferred is the PARI inhalation nebulizerdescribed in PCT/US00/29541 modified to meet the requirements of thisinvention.

a. Dry Powder Inhalers

Dry powder is administered as such using devices which deliver the drypowder directly to the lungs.

There are two major designs of dry powder inhalers. One design is themetering device in which a reservoir for the drug is placed within thedevice and the patient adds a dose of the drug into the inhalationchamber. The second is a factory-metered device in which each individualdose has been manufactured in a separate container. Both systems dependupon the formulation of drug into small particles of mass mediandiameters from 1 to 5 microns, and usually involve co-formulation withlarger excipient particles (typically 100 micron diameter lactoseparticles). Drug powder is placed into the inhalation chamber (either bydevice metering or by breakage of a factory-metered dosage) and theinspiratory flow of the patient accelerates the powder out of the deviceand into the oral cavity. Non-laminar flow characteristics of the powderpath cause the excipient-drug aggregates to decompose, and the mass ofthe large excipient particles causes their impaction at the back of thethroat, while the smaller drug particles are deposited deep in thelungs.

Current technology for dry powder inhalers is such that payload limitsare around 100 mg of powder. The lack of long-term stability ofaztreonam in an aqueous solution due to hydrolysis allows dry powderinhaler technology to become a preferred delivery vehicle for aztreonamdry powder.

C. Aerosol or Dry Powder Particle Size

Particle size of the aztreonam aerosol formulation is one of the mostimportant aspect of the invention. If the particle size is larger than5μ then the particles are deposited in upper airways. If the particlesize of the aerosol is smaller the 1μ then it does not get deposited inthe endobronchial space but continues to be delivered into the alveoliand may get transferred into the systemic blood circulation.

A jet nebulizer utilizes air pressure to break a liquid solution intoaerosol droplets. An ultrasonic nebulizer works by a piezoelectriccrystal that shears a liquid into small aerosol droplets. A pressurizednebulization system forces solution under pressure through small poresto generate aerosol droplets. A vibrating porous plate device utilizesrapid vibration to shear a stream of liquid into appropriate dropletsizes. However, only some formulations of aztreonam can be efficientlynebulized as the devices are sensitive to pH and salinity.

In dry powder inhalers, the aztreonam dry powder prepared as describedabove in dosages from 1-100 mg, preferably from 10-50 mg of dry powderas particles having sizes between 1 and 5μ, is used directly.

D. Efficacy of Aztreonam Nebulization

Selection and choice of the nebulizer greatly effects efficacy of theinhalable aztreonam delivery.

A combination of an aerosol formulation of aztreonam and a nebulizingdevice significantly enhance the efficiency and speed of drugadministration. Currently, for example the average time foradministration of other aerosolized drugs, such as for exampletobramycin, is 15-20 minutes per dose. The time required for thistreatment represents a significant burden to the patient and contributeto reduced compliance with the BID regimen.

Furthermore, the nebulizer system used for tobramycin administration isless efficient than new atomizing devices. The total deposited dose oftobramycin in the lung is in the 12 to 15% range. Approximately 30% ofthe dispensed drug remains in the nebulizer at the end of treatment, andof the portion that is aerosolized, about 30% is emitted as particlestoo large or small to reach the lower airways.

The novel atomizing nebulizer, with an output of 8 to 10microliters/seconds, or 0.48 to 0.60 ml/minute, is capable of deliveringdrug material 2 to 4 times faster than the prior nebulizers exemplarizedby PARI LC plus nebulizer. Furthermore, the novel nebulizer is able toaerosolize approximately 90% of the dispensed dose, with 85% or more ofthe aerosol particles being within the size range required for lowerairway deposition. As a result, administration of a specificallydesigned formulation of aztreonam using the atomizing nebulizer leads tosubstantial improvement in local delivery to the airways, to a shortertime required for delivery and, depending on the final concentration ofaztreonam solution, reduces treatment time to as little as four minutes.

VI. Treatment of Pulmonary Bacterial Infections

This invention provides an efficacious treatment and prevention of acuteand chronic pulmonary bacterial infections caused by Pseudomonasaeruginosa, Escherichia coli, Klebsiella pneumoniae, Pseudomonasaeruginosa, Haemophilus influenzae, Proteus mirabilis, Enterobacterspecies and Serratia marcescens, as well as infection caused byantibiotic resistant strains Burkholderia cepacia, Stenotrophomonasmaltophilia, Alcaligenes xylosoxidans, and multidrug resistantPseudomonas aeruginosa.

A. Two Modes of Inhalable Treatment

A method for treatment of pulmonary infections comprises administrationof aztreonam in inhalable form whether by aerosol or as a dry powder,several times a day. The aztreonam daily dose is between 1 and 500mg/day, with exceptional dose up to 750 mg/day administered in from 1-50mg/ml for aerosol and from 2 to 200 mg daily dose of dry powderadministered in a dose of 1-100 mg/one treatment. The aztreonam dosageand dosing frequency depends on the type of bacterial infection,severity thereof, age of the patient, the conditions of the patient,etc. In case of cystic fibrosis patients where the lung air capacity isdiminished, the dosing is more frequent with lower doses.

The dry powder formulation suitable for treatment of pulmonaryinfections comprises 1 to 200 mg, preferably about 10 to 100 mg, ofpowder in an amorphous or crystalline state in particle sizes between 1and 5 microns in mass median average diameter necessary for efficaciousdelivery of aztreonam into the endobronchial space. The dry powderformulation is delivered one to four or more times daily, preferablytwice daily. The dry powder formulation is temperature stable and has aphysiologically acceptable pH of 4.5-7.5, preferably 5.5 to 7.0, and anover five year long shelf life.

B. Treatment of Infections in Patients with Suppurative PulmonaryDiseases

Aerosol therapy of this invention is particularly useful for treatmentof patients suffering from suppurative pulmonary diseases and isespecially suitable for treatment of patients with cystic fibrosis,bronchiectasis and those patients on the mechanical ventilation.

Previously, aerosol therapy for cystic fibrosis inhaled (ATCF)antibiotics have demonstrated significant benefit of such treatment tocystic fibrosis (CF) patients suffering from chronic pulmonaryinfections.

In the US, the most widely used and successful agent in this regard hasbeen tobramycin, which has been shown to produce substantialimprovements in lung function and other clinical parameters.

It has now been discovered that inhalable aztreonam provides successfultreatment in cystic fibrosis, bronchiectasis or other suppurativepulmonary disease for pulmonary infections caused by gram-negativebacteria and particularly those caused by antibiotic resistantBurkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenesxylosoxidans and multidrug resistant Pseudomonas aeruginosa.

Treatment of these multi-resistant bacterial infections with aerosolizedaztreonam has been successful in eradication of the bacteria asdescribed in Example 2.

Such treatment is stand alone or may be complementary treatment to otherantibiotics, such as tobramycin, which upon extended use, results in thedevelopment of anti-tobramycin resistance. When the treatment withtobramycin is interspaced with periods of treatment with aztreonam, suchresistance either does not develop or recedes.

C. Limitations of Current Aerosolized Antibiotics in Treatment of CysticFibrosis

To date, an aminoglycoside tobramycin is the only antibiotic with FDAapproval for administration as an aerosol. However, despite the benefitsobtained in cystic fibrosis patients with administration of aerosolizedtobramycin, its utility is somewhat limited.

First, frequent use of aminoglycosides to control pulmonaryexacerbations leads to selective development of resistant Pseudomonasaeruginosa strains. The widespread emergence of such organisms isacknowledged as a growing crisis in the CF community. For example, 21%of patients screened from 69 different CF centers for the phase IIItobramycin clinical trials had isolates resistant to tobramycin (MIC>16μg/mL). Accordingly, many clinicians are reluctant to prescribe thisaerosolized aminoglycoside as chronic suppressive therapy, fearing thatit could further promote resistance and thus diminish the effectivenessof IV therapy. In order to reduce the risk of such treatment-emergentresistance, tobramycin therapy is restricted to cycles of 28 days on and28 days off the drug.

A second limitation of aerosolized tobramycin is its lack of activityagainst several intrinsically tobramycin resistant bacteria, includingStenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Burkholderiacepacia, the latter of which is widely recognized as a significantthreat to cystic fibrosis patients. Cystic fibrosis patients infectedwith Burkholderia cepacia have an increased rate of mortality, and manyexperience a rapid fatal course, as described in Am. J. Respir. Crit.Care Med., 160:1572-1577, (1999). Additionally, Burkholderia cepacia isa transmittable infection which can cause epidemic spread among cysticfibrosis patients. Therefore, a patient infected with Burkholderiacepacia must be isolated from other patients.

Aerosolized aztreonam does not induce resistance to aminoglycosides andhas activity against resistant pathogens observed in cystic fibrosispatients.

An aerosolized aztreonam can either replace tobramycin, or be used as analternative and intermittent treatment for tobramycin during the 28-daytobramycin free periods, which are required to prevent development ofpermanent resistence to tobramycin.

Aztreonam is an antibiotic with excellent activity against many aerobicgram-negative bacteria, including multi-resistant Pseudomonasaeruginosa. The spectrum of activity of aztreonam is similar to that ofthe aminoglycoside antibiotics tobramycin and gentamycin, and itsantipseudomonal activity is comparable to ceftazidine.

Aztreonam resists destruction by most bacterial β-lactamases, which arethe source of much treatment-emergent resistance to β-lactam antibioticsfrequently appearing among hospitalized patients.

Aztreonam's activity against gram-negative bacteria, especiallyPseudomonas aeruginosa, combined with its excellent safety profile makesit a good alternative to aminoglycosides in the treatment of chronicpulmonary infections among cystic fibrosis patients. Thus far, clinicaluse of aztreonam in CF patients has included IV administration ofaztreonam as single agent therapy or in combination with otherantibiotics for treatment of pulmonary exacerbations.

D. Advantages of Aztreonam as an Aerosolized Antibiotic

Aztreonam possesses several features that make it very attractive foraerosol administration to CF patients.

The first of these features stems from its mechanism of action, which,unlike aminoglycoside antibiotics, involves preferential binding topenicillin binding protein 3 (PBP3) and subsequent interference withbacterial cell wall synthesis. Because aztreonam's mechanism of actiondiffers from that of tobramycin, its use does not contribute toemergence of aminoglycoside-resistant strains of Pseudomonas aeruginosa.

The second advantage of an aerosolized formulation of aztreonam is itsactivity against tobramycin resistant, and multidrug resistantPseudomonas aeruginosa. When isolates from patients enrolled in thePhase II tobramycin trials were examined, nearly 75% of isolates with atobramycin MIC>16 μg/mL were susceptible to aztreonam.

The third feature is aerosolized aztreonam ability to controlintrinsically tobramycin resistant organisms, especially Burkholderiacepacia, which is considered resistant to the levels of aztreonamachieved by parenteral administration.

VII. In Vitro Testing

In order to test antibacterial activity of aerosolized aztreonam againstmulti-resistant strains of Pseudomonas aeruginosa, Burkholderia cepacia,Stenotrophomonas maltophilia and Alcaligenes xylosoxidans, the in vitroactivities of aztreonam in concentrations corresponding to thoseachievable with inhalable aztreonam were tested against clinicalisolates from cystic fibrosis patients.

The aztreonam aerosol delivery according to the invention achievesconcentrations of aztreonam to reach levels from 500 to as high as 8000μg/ml, with an average level around 2,000 μg/ml, of aztreonam in thesputum. These levels depend on the formulation as well as on thenebulizer used for aerosolization. With certain nebulizers theconcentration of aztreonam can reach an average level of 5,000 μg/ml.

In vitro determined susceptibilities of the tested bacteria ispredictive of clinical efficacy of inhaled aztreonam aerosol or drypowder.

Aztreonam kills by lysing cell walls as long as the local concentrationof antibiotic exceeds the bacteria minimal inhibitory concentration(Med. Clinics N. Am., 79: 4, 733-743, (1995)).

The in vitro activity of high aztreonam concentrations against clinicalisolates of B. cepacia, S. maltophilia and A. xylosoxidans was tested atthe Children's Hospital and Regional Medical Center in Seattle, Wash.Testing was performed on broth microdilution trays made with 2 foldconcentrations of aztreonam from 2 to 2048 μg/mL. Staphylococcus aureus,a gram positive organism, was used as a negative control. Detailedprocedure used for testing is described in Example 1. Results are seenin Table 1. TABLE 1 Organism (# of isolates) MIC Range MIC50 MIC90 P.aeruginosa (54) 2-1024 16 512 B. cepacia (38) 2-2048 32 512 S.mallophilia (20)  8->2048 256 >2048 A. xylosoxidans (20) 2 > 2048 2562048 S. aureus (20) 512-2048  1024 2048

For testing, each microwell plate contained a 2-fold dilution, 2, 4, 8,16, 32, 64, 128, 256, 512, 1024 and 2048 of aztreonam. Each platecontaining the microwells was used to test one isolate of one organism.

Table shows the different species of bacteria tested for sensitivity,that is the ability of the antibiotic to inhibit its growth, toaztreonam, with the number of isolates for each species given inparenthesis. The column designated “MIC range” shows the range of thelower and upper limits of sensitivities seen in the tested isolates. Thecolumn designated MIC50 shows the median level of sensitivity for themost sensitive 50% isolates. The final column, designated MIC90, showsthe median value for the level of sensitivity for the most sensitive 90%of the isolates.

Table 1 shows results of comparative in vitro activity of aztreonamagainst clinical isolates obtained from cystic fibrosis patients.

For interpretation of this data, these values which represent whatconcentration of aztreonam is required to inhibit growth of bacteria arecompared with the concentrations of aztreonam obtainable by thedifferent routes of administration. Thus, for intravenous administrationof aztreonam, the serum level following administration of 2 g ofaztreonam, the maximum allowed intravenous dose, the serum level is peakis 256 μg/ml and then declines rapidly. At six hours following theadministration, the aztreonam level in the serum is in the range of 16μg/ml. For safety reasons, intravenous aztreonam can only beadministered every six hours. With the possible exception of Pseudomonasaeruginosa that has a MIC50 of 16 μg/ml, all other organisms would bepredominantly resistant to intravenous aztreonam, as their level ofresistance exceeds even the peak concentration (256 μg/ml) of serumconcentration of sputum of aztreonam following intravenousadministration. Since, however, the bacteria resistance is relative todrug concentration, for aerosol administration, the peak concentrationshould be at least in the 500 to 2000 μg/ml range. Such range isachieved with the doses of aztreonam and the formulation of theinvention combined with the efficient nebulizer, according to thisinvention. At the 500-2000 μg/ml concentration in the sputum, theaerosol therapy according to this invention is able to treat mostendobronchial infections caused by gram-negative bacteria, specificallythose bacteria listed in Table 1, with exception, of course, ofStaphyloccocus aurelius.

The MIC50 and MIC90 have shown that treatment of P. aeruginosa withinhalable aztreonam eradicates most P. aeruginosa isolates with the highconcentrations of aztreonam in sputum of cystic fibrosis patientsobtainable after aerosol delivery. The data obtained for Burkholderiacepacia isolate indicated that at least half of patients would beexpected to respond to such treatment with eradication of the bacteria.If sufficiently high concentrations of aztreonam are delivered to thelung, the percentage is expected to be higher. Since the Burkholderiacepacia infection is now viewed as a largely untreatable condition,treatment with inhalable aztreonam by aerosol is the first documentedefficacious therapy.

The results obtained in these studies are surprising and unexpected asthere is no indication in the literature that Burkholderia cepacia issusceptible to treatment with aztreonam. The data also shows that someisolates of S. maltophilia and A. xyloxidans respond to highconcentration of aztreonam.

Inhalation of aztreonam according to the invention permits reachingconcentrations of aztreonam in the sputum as high as 2000-5,000 μ/mL.The sputum aztreonam levels achieved via aerosol administration exceedthose required to inhibit organisms responsible for otherwiseuntreatable infections in CF patients.

Furthermore, aztreonam delivered by inhalation to all patients withBurkholderia cepacia and/or S. maltophilia and/or A. xyloxidans togetherwith other antibiotics whether administered systemically parenterally orby inhalation contributes to synergy of such treatment. A combination ofinhalable aztreonam with other antibiotics provides another therapeuticapproach to treat multi-resistant bacterial strains.

The studies described herein demonstrated that the concentrations ofaztreonam achieved following aerosol administration have activityagainst Burkholderia cepacia isolated from CF patients' sputum as wellas against other bacteria which are largely resistant to treatment withother antibiotics.

The MIC50 and MIC90 observed for a gram positive bacteria,Staphylococcus aureus, show that high concentrations of aztreonam hadsome activity against this gram positive bacteria. These findings,however, have no great significance as there are many other drugs withreasonable efficacy against Staphyloccocus aureus.

VII. In Vivo Testing

The infections requiring particular attention are infections caused byand include B. cepacia, S. maltophilia and A. xylosoxidans, as well asmulti-resistant strains of Pseudomonas aeruginosa, the most clinicalsignificant infection is the former.

In order to determine if an appropriately formulated aztreonam foraerosolization could become effective for treatment of these rare butvery resistant bacterial strains, the treatment with aerosolizedaztreonam was initiated and tested in a cystic fibrosis patient having asevere Burkholderia cepacia infection which did not respond to anytreatment. The clinical treatment and results obtained with anaerosolized aztreonam is described in Example 2.

Utility

The method of treatment and the inhalable aztreonam compositionsdisclosed herein is suitable for treatment of respiratory tractinfections caused by Burkholderia cepacia, Stenotrophomonas maltophilia,Alcaligenes xylosoxidans, and multidrug resistant Pseudomonas aeruginosaas well as for treatment of other pulmonary infections caused bygram-negative bacteria.

EXAMPLE 1 In Vitro Testing of Isolates from Cystic Fibrosis Patients

This example describes procedure used for in vitro studies of bacterialisolates obtained from cystic fibrosis patients.

Bacterial respiratory tract isolates (144) from patients with CF thathad been stored at −70° C. were cultivated by two consecutive overnightpassages at 37° C. on 5% blood agar (Remel, Lenexa, Kans.).

Minimal inhibitory concentrations (MIC's) were determined by thefollowing steps:

MIC Antimicrobial Testing Aerobic Organisms

1. MIC trays were brought to room temperature.

2. 3.0 ml physiological saline was inoculated with an 18-24 h culture oforganism to be tested to a turbidity equal to a 0.5 McFarland Standard(1.5×10⁸ CFU/ml). This corresponds to an OD600 of 80-88% transmission.

3. Within 15 minutes of preparation, the adjusted inoculum suspensionwas diluted by transferring 100 ml into a 2.9 ml diluent of sterilewater.

4. The suspension was gently mixed by inversion and 10 ml was dispensedinto each MIC well having initial volume of 100 μl. The finalconcentration in each well was equal to 5×10⁵ CFU/ml or 5×10⁴ CFU/well.

5. Trays were incubated aerobically at 37° C. for 16-20 hours. The sameincubation temperature was maintained for all cultures. Microdilutiontrays were not stacked more than four high.

6. Antimicrobial endpoint was read and recorded as the first wellshowing no readily visible growth or haze as detected by the unaidedeye.

7. The microdilution trays were contacted with 2 fold concentrations ofaztreonam from 2 to 2048 mg/mL. Each microwell plate was treated with a2-fold dilution of aztreonam in following amounts: 2, 4, 8, 16, 32, 64,128, 256, 512, 1024 and 2048 μg/ml. Each plate containing the microwellswas used to test one isolate of one organism.

8. Results were read and recorded.

EXAMPLE 2 Clinical Treatment of Patient with Burkholderia cepacia

This example describes a first finding of efficacy of the aerosolizedaztreonam treatment of a cystic fibrosis patient suffering fromresistant Burkholderia cepacia.

The patient was a 20-year-old female with cystic fibrosis and end stagelung disease. She had been diagnosed with Burkholderia cepacia pulmonaryinfections that had become resistant to all known intravenous, oral andinhaled antibiotics. She had two-documented genetically differentstrains of Burkholderia cepacia. For this reason the patient wasrejected as a candidate for a lung transplant.

The patient was provided with a formulation of the invention comprising200 mg/ml of aztreonam and instructed to use this formulation in 3 to 5ml of diluent and use it in an air compressor powered breath enhancedjet nebulizer and take the therapy twice a day. This type of nebulizeronly delivers about 10 to 20% of the dose placed in the nebulizers tothe lungs, however, that was only nebulizer available to the patient forhome treatment.

After three months of continuous twice a day therapy, the pulmonaryinfection was successfully treated and no evidence of Burkholderiacepacia could be detected. The patient was considered treated from theinfection and eventually underwent a successful lung transplantprocedure.

There was no postoperative reoccurrence or relapse of the Burkholderiacepacia infection despite of intensive immunosuppression therapyfollowing the transplantation.

These findings were surprising since previous use of commerciallyavailable aztreonam in an older generation delivered in even lessefficient nebulizers did not lead to eradication of P. aeruginosa asdescribed in Clinics Chest Med., 19:473-86, (September 1998). In thistrial, the authors stopped therapy at the development of any aztreonamresistance rather than continuing treating these patients. Prior workdid not test or speculate that this therapy could be effective intreating other gram negative bacteria including Burkholderia cepacia, S.maltophilia, X. xylosoxidans, or other multidrug resistant pseudomonasinfections.

The results obtained with treatment of the above patient are even moresurprising in that the eradication of Burkholderia cepacia is extremelyrare occurrence, particularly when the infection is well established aswas in the case of this patient.

EXAMPLE 3 Preparation of Aztreonam Dry Powder

This example provide methods and procedures used for preparation ofaztreonam containing inhalable dry powder.

For dry powder formulation of the invention, a purified aztreonamantibiotic, or a pharmaceutically acceptable salt thereof, is milled toa powder having mass median average diameters ranging from 1 to 5μ bymedia milling, jet milling, spray drying, or particle precipitationtechniques.

Particle size determinations is made using a multi-stage Andersoncascade impactor.

Media milling may be accomplished by placing the drug substance into amill containing, for example, stainless steel or ceramic balls androtating or tumbling the material until the desired drug particle sizeranges are achieved.

Jet milling uses very high pressure air streams to collide particleswith one another, with fine particles of the desired size beingrecovered from the mill.

Spray drying is achieved by spraying a fine mist of drug solution onto asupport and drying the particles. The particles are then collected.

Particle precipitation is achieved by adding a co-solvent to spray driedparticles. The solubility of the drug falls to the point where soliddrug particles are formed. The particles are collected by filtration orcentrifugation. Precipitation has the advantage of being highlyreproducible and can be performed under low temperature conditions,which reduce degradation.

EXAMPLE 4 Dry Powder Inhalators

The dry powder formulations of the invention may be used directly inmetered dose or dry powder inhalers.

A metered dose inhaler consists of three components: a canistercontaining the propellant drug suspension, a metering valve designed todeliver accurately metered volumes of the propellant suspension, and anoral adapter which contains a spray orifice from which the metered doseis delivered. In the rest position, the metering chamber of the valve isconnected to the drug suspension reservoir via a filling groove ororifice. On depression of the valve this filling groove is sealed andthe metering chamber is exposed to atmospheric pressure via the sprayorifice in the oral adapter and the valve stem orifice. This rapidpressure reduction leads to flash boiling of the propellant andexpulsion of the rapidly expanding mixture from the metering chamber.The liquid/vapor mixture then enters the expansion chamber which isconstituted by the internal volume of the valve stem and the oraladapter. The mixture undergoes further expansion before being expelled,under its own pressure, from the spray nozzle. On exit from the sprayorifice, the liquid ligaments which are embedded in propellant vapor aretorn apart by aerodynamic forces. Typically, at this stage, the dropletsare 20 to 30μ in diameter and are moving at the velocity of sound of thetwo-phase vapor liquid mixture (approximately 30 meters per second). Asthe cloud of droplets moves away from the spray nozzle, it entrains airfrom the surroundings and decelerates, while the propellant evaporatesthrough evaporation, the entrained droplets eventually reach theirresidual diameter.

At this point, the particles/droplets consist of a powdered drug corecoated with surfactant. Depending on the concentration and the size ofthe suspended material the powdered drug core consists of eitherindividual drug particles or aggregates. Currently, meter dose inhalertechnology is optimized to deliver masses of 80 to 100 micrograms ofdrug, with an upper limitation of 1 mg of drug deliverable.

An alternated route of dry powder delivery is by dry powder inhalers.There are two major designs of dry powder inhalers, device-meteringdesigns in which a reservoir of drug is stored within the device and thepatient “loads” a dose of the device into the inhalation chamber, andfactory-metered devices in which each individual dose has beenmanufactured in a separate container. Both systems depend upon theformulation of drug into small particles of mass median diameters from 1to 5 microns, and usually involve co-formulation with large excipientparticles (typically 100 micron diameter lactose particles). Drug powderis supplied into the inhalation chamber (either by device metering or bybreakage of a factory-metering dosage) and the inspiratory flow of thepatient accelerates the powder out of the device and into the oralcavity. Non-laminar flow characteristics of the powder path cause theexcipient-drug aggregate to decompose, and the mass of the largeexcipient particles causes their impaction at the back of the throat,while the inhaler drug particles are deposited deep in the lungs.Current technology for dry powder inhalers is such that payload limitsare around 50 mg of powder (of which drug is usually a partial componentby mass). Excipients commonly used are lactose, however in the case ofaztreonam free base the addition of the amino acids lysine or leucinewill lead to better powder formation.

Effective dosage levels of aztreonam antibiotic for dry powderinhalation and metered dose inhalation result in the delivery of atleast about 25 mg, and more preferable about 50 to about 100 mg ofaztreonam antibiotic compound to the lung of the patient receivingtreatment. Depending on the efficiency of the dry powder deliverydevice, dry powder formulations suitable for use in the inventioncomprise from about 1.0 to about 250 mg, preferably from about 10 toabout 100 mg of powder in an amorphous or crystalline state in particlesizes between 1 and 5 microns in mass median average diameter necessaryfor efficacious delivery of the antibiotic into the endobronchial space.The dry powder formulation may be delivered from 1 to 4 times daily,preferably twice daily, for a period of at least one day, morepreferably at least 5 days and most preferably at least fourteen days orlonger. The dry powder formulations are temperature stable and have aphysiologically acceptable pH of 4.0 to 7.5, preferably 5.5 to 7.0, andlong shelf lives.

EXAMPLE 5 Preparation of Aztreonam Sodium Salt

This example describes procedure used for preparation of aztreonamsodium salt.

To a solution of 10 g (23 mmol) of aztreonam in 100 mL of MeOH cooled inan ice bath was added dropwise 23 mL (23 mmol, 1.0 eq) of 1N sodiumhydroxide solution. The resulting solution was warmed to ambienttemperature over a period of 30 min, and then the solvent was removedunder reduced pressure. Diethylether (50 mL) was added and the slurryconcentrated. This step was repeated four times to provide a yield of10.1 g (96%) of aztreonam sodium salt as a white powder.

EXAMPLE 6 Preparation of Aztreonam Sodium Salt Solutions

This example describes procedure used for preparation of aztreonamsodium salt solution.

Aztreonam (10 g, 23 mM) free base was added to a tared 100 mL Erlenmeyerflask. Methanol (25 mL) was added to the flask with agitation bymagnetic stirrer. 1N sodium hydroxide (23 mL, 1 equivalent) wasgradually added while stirring. When solution was clear, it was removedfrom stir plate and the excess solvents were removed under reducedpressure to give a dry solid. Deionized water (166 mL) was addeddropwise to the dry solid and the pH of the resulting solution wasadjusted to the desired value of 6.5 by dropwise addition of 1N sulfuricacid while monitoring with a pH meter. The above procedure was used toprepare aztreonam salt solutions at 60 mg/mL by adjusting the weight ofaztreonam and the volume of 1N sodium hydroxide.

EXAMPLE 7 Aztreonam Formulation

This example illustrates preparation of the aztreonam containingformulation of the invention.

1. Hot water for injection (WFI) was thoroughly flushed through 20 LMillipore product vessel.

2. Aztreonam potency (g/L) was assayed, and its efficacy determined.

3. Aztreonam was added to a wide mouth specimen bottle and label ofproduct vessel in the accurately weighed amount.

4. 11.25 kg of WFI was dispersed into a clean 20 L Millipore productvessel.

5. With moderate agitation, 33.75 g sodium chloride, USP, was slowlyadded and mixed until dissolved.

6. WFI was added to the product vessel to 12 kg and mixed for 5 minutes.

7. With continual mixing, 100 mL 5N of sulfuric acid (H₂SO₄) wascarefully added for each liter of WFI in the final formulation.

8. Product vessel was sparged with nitrogen (N₂).

9. After approximately 15 minutes of sparging, dissolved oxygen (O₂) wasmeasured by continuous monitoring of dissolved oxygen in the tank, usinga probe.

10. Measuring of dissolved O₂ was continued until five (5) consecutivemeasurements ≦3 ppm dissolved O₂.

11. With continuous sparging of N₂ and moderate mixing, the 562.5 g (50g/L) aztreonam was added and mixed until dissolved.

12. 20 mL sample from product formulation was removed and pH wasmeasured. Product formulation was adjusted to final pH value of 6.0.

13. An aliquot of product formula was sampled and analyzed for aztreonamconcentration.

14. An aliquot of product formula was analyzed for pH.

15. An aliquot of product formula was analyzed for dissolved O₂ (intriplicate).

16. When the batch met quality control testing criteria, the product wasreleased for use.

17. The product was frozen to −20° C. and kept at this temperature orbelow until the actual use.

EXAMPLE 8 Testing Nebulizers

A clinical study is conducted in order to determine the concentration ofaztreonam in the aerosol formulation required to achieve a sputumconcentration between 500 μg/gm and 2000 μg/gm sputum at 10 minpost-completion of aerosol administration using an atomizing, ultrasonicor jet nebulizer.

In this study, cystic fibrosis patients receive serial doses of 250 mgaztreonam (5 ml of a 50 mg/ml solution in ¼ NS) from each of thenebulizers. The doses are separated by at least 2 days and not more than5 days. Peak serum and sputum concentrations are assessed.

EXAMPLE 9 Clinical Trial Protocol

This example describes a protocol used for clinical trial and to comparethe pharmacokinetics of increasing dosage of an aztreonam formulationadministered by the PARI electronic nebulizer to patients with cysticfibrosis.

The primary aim of this study is to determine which of the tested doselevels delivered by aerosol can deliver sufficient amount of aztreonamor a salt thereof to achieve a mean peak sputum aztreonam concentrationof 2000 μg/gm or greater measured 10 minutes after the completion ofnebulization in patients with CF.

The secondary aim is to determine whether the aztreonam concentrationrequired to achieve a mean peak sputum concentration of 2000 μg/gm orgreater is safe and well tolerated by the patient.

Study Design

This is an open label, multicenter, randomized, dose escalation study.

Each arm is different dose. Two arms deliver the same aztreonamformulation.

1. 0.5 ml of aztreonam solution of 50 mg/ml

2. 1.0 ml of aztreonam solution of 50 mg/ml

3. 2.0 ml of aztreonam solution of 50 mg/ml

4. 3.0 ml of aztreonam solution of 50 mg/ml

5. 4.0 ml of aztreonam solution of 50 mg/ml

6. 5.0 ml of aztreonam solution of 50 mg/ml

Efficacy and Safety Assessment

In this study, the following efficacy and safety parameters areassessed:

The efficacy is determined for each nebulizer by measuring concentrationof aztreonam in sputum 10 minutes after completion of nebulization. Meanconcentration of 2000 μg/gm of sputum is considered adequate.

The safety parameters assessed:

1. Incidence of treatment related adverse reactions occurring during theadministration of the aerosolized aztreonam at the different doselevels.

2. Acute bronchospasm at the time of drug administration.

3. Absorption of aztreonam into the systemic circulation.

Each patient receives in random order at least one administration. Eachaerosol administration is separated by a minimum of 48 hr. Sputumsamples are collected at baseline, 1, 2, 4 and 6 hours post-completionof the aerosol drug administration to measure aztreonam concentration.Serum samples are collected at baseline, 1, 2, 4 and 6 hourspost-completion of aerosol administration to measure aztreonam levels.

Airway irritation and acute bronchospasm are assessed by measuringspirometry immediately prior to and 30 min post-completion of aerosoladministration. A decrease in forced expired volume in one second(FEV1) >15% in the 30 min spirometry test is considered evidence ofbronchospasm.

The primary objective of this study is to determine if and at what dosethe PARI electronic nebulizer tested can aerosolize sufficient aztreonamsulfate to achieve a mean peak sputum aztreonam concentration of 2000μg/gm or greater in at least 85% of patients with CF measured 10 minutesafter the completion of nebulization.

The second objective is to determine whether the aztreonam concentrationrequired to achieve a mean peak sputum concentration of 2000 μg/gm orgreater is safe and well tolerated by the patient. Safety is defined asa lack of acute bronchospasm and minimal systemic absorption.

Patient Treatment

All patients with underlying disease of cystic fibrosis (CF), confirmedat entry by the inclusion/exclusion criteria specified in this protocol,are eligible for enrollment into the study. Investigators at theparticipating CF centers select patients that meet all of the inclusioncriteria and one of the exclusion criteria.

Eligible patients are admitted to the study center on the day of thestudy and receive aerosol therapy if they fulfilled entrance criteria.

Physical exam is administered by a physician or RC nurse prior toinitial aerosol treatment only.

Vital signs, height, weight, oximetry, assessment of current respiratorystatus and brief medical history are used.

Sputum and serum samples are collected to measure baseline aztreonamconcentrations.

Patients are sitting upright and use nose clips during the aerosoladministration.

The total duration of time and the number of inhalations required tocomplete the aerosol treatment are recorded.

Any evidence of wheezing or respiratory distress are recorded as well asnumber of rest periods required by the subject because of dyspnea orexcessive coughing during the administration period.

Immediately after completing the aerosol therapy, the subject rinse with30 ml of normal saline through the mount, gargled for 5-10 seconds andexpectorated the rinse. This is repeated for a total of three rinses.

Sputum specimens are collected at 10 minutes after rinsing oral cavityand 2 hours after completion of the aerosol drug administration.

Serum is collected at 1 and 2 hours after completion of the aerosol drugadministration for determination of the aztreonam levels.

Spirometry is obtained 30 minutes following completion of the aerosoldrug administration.

Following the last aerosol treatment of the study, patients receive abrief physical exam after post-spirometry has been measured.

1. A method of treating pulmonary infections caused by gram-negativebacteria, said method comprising delivering an aerosol of an inhalableaztreonam formulation to the lung endobronchial space of airways of apatient in need thereof, wherein said inhalable aztreonam formulationdoes not comprise an arginine salt of aztreonam.
 2. The method of claim1 wherein said inhalable aztreonam formulation comprises from about 1 toabout 250 mg of aztreonam or a pharmaceutically acceptable salt thereofper one dose.
 3. The method of claim 1 wherein said aerosol comprisesfrom about 1 to about 250 mg of aztreonam or a pharmaceuticallyacceptable salt thereof dissolved in about 1 to about 5 ml of a salinesolution containing from about 0.1 to about 0.9% of sodium chloride oran equivalent thereof.
 4. The method of claim 3 wherein said aerosol isnebulized into particles, at least 70% of which have sizes between about1 and about 5 μm.
 5. The method of claim 4 wherein the inhalableaztreonam formulation is delivered one to twelve times a day, providedthat a total dose of aztreonam or a pharmaceutically acceptable saltthereof is not higher than 750 mg a day.
 6. The method of claim 5wherein the aerosol is delivered by a nebulizer.
 7. The method of claim6 wherein the nebulizer is an atomizing nebulizer or a jet, ultrasonic,electronic or vibrating porous plate nebulizer, wherein said nebulizeris able to or is modified to be able to nebulize volume from about 1 toabout 5 ml of the inhalable aztreonam formulation to particle, the sizesof which range from about 1 to about 5 μm.
 8. The method of claim 7administering the inhalable aztreonam formulation comprising from about10 to about 200 mg of the aztreonam or a pharmaceutically acceptablesalt thereof.
 9. The method of claim 8 comprising administering theinhalable aztreonam formulation comprising from about 50 to about 100 mgof aztreonam or a pharmaceutically acceptable salt thereof.
 10. Themethod of claim 9 wherein the aerosol has pH from about 4.5 and about7.5 and the saline solution contains from about 0.1 to about 0.45% ofsodium chloride.
 11. The method of claim 10 wherein said aerosol has pHfrom about 4.5 to about 5.5.
 12. The method of claim 3 comprisingadministering the aerosol comprising from about 10 to about 100 mg ofthe aztreonam or a pharmaceutically acceptable salt thereof once, twice,three or four times a day.
 13. A method of treating infections caused bythe gram-negative bacteria in patients with cystic fibrosis orbronchiectasis, or in patients on ventilators with pneumonia, comprisingdelivering an aerosol of an inhalable aztreonam formulation to the lungendobronchial space of airways of a patient in need thereof, wherein theinhalable aztreonam formulation comprises from about 1 to about 250 mgof aztreonam or a pharmaceutically acceptable salt thereof per one dose,wherein said pharmaceutically acceptable salt is selected from the groupconsisting of acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, bytyrate, camphorate, camphorsulfonate,citrate, digluconate, cyclopentanepropionate, dodecylsulfate,ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, lysinate, maleate,methanesulfonate, nicotinate, 2-napthalene-sulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate andundecanoate.
 14. An inhalable pharmaceutically acceptable compositioncomprising from about 1 to about 250 mg per one dose of aztreonam or apharmaceutically acceptable salt thereof, said composition suitable fortreatment of pulmonary bacterial infections caused by gram-negativebacteria Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenesxylosoxidans or multidrug resistant Pseudomonas aeruginosa, wherein saidaztreonam or the pharmaceutically acceptable salt thereof is prepared asan aerosolable saline solution.
 15. The composition of claim 14comprising from about 1 to about 250 mg or aztreonam base or thepharmaceutically acceptable salt thereof per one dose administered once,two, three or four times a day with a proviso that when the compositionis administered more than three times a day, the total dose of theaztreonam or the pharmaceutically acceptable salt thereof does notexceed 750 mg a day.
 16. The composition of claim 15 wherein saidpharmaceutically acceptable salt is selected from the group consistingof acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, bytyrate, camphorate, camphorsulfonate, citrate, digluconate,cyclopentanepropionate, dodecylsulfate, ethanesulfonate,glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, lysinate, maleate, methanesulfonate,nicotinate, 2-napthalenesulfonate, oxalate, pamoate, pectinate,persulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate.17. The composition of claim 16 wherein said aztreonam or thepharmaceutically acceptable salt thereof is dissolved in from about 1 toabout 5 ml of the aerosolable solution comprising at least 0.09% ofsodium chloride or equivalent amount of bromine, iodine or bicarbonatesalt.
 18. The composition of claim 17 wherein said aerosolable salinesolution comprises from about 0.1 to about 0.45% of sodium chloride andhas a pH from about pH 4.5 to about pH 7.5.
 19. The composition of claim18 wherein said pH is from about pH 4.5 to about pH 5.5.
 20. Thecomposition of claim 19 nebulized into an aerosol having a particle sizewith a mass medium average diameter from about 1 to about 5μ.
 21. Anaerosol composition comprising from about 1 to about 250 mg of aztreonamor a pharmaceutically acceptable salt thereof selected from the groupconsisting of acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, bytyrate, camphorate, camphorsulfonate,citrate, digluconate, cyclopentanepropionate, dodecylsulfate,ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, lysinate, maleate,methanesulfonate, nicotinate,2-napthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate andundecanoate, said aztreonam or the salt dissolved in from about 1 toabout 5 ml of a solution containing from about 0.1 to about 0.9% sodiumchloride.
 22. A method of treating pulmonary infections caused bygram-negative bacteria comprising delivering an aerosol of an inhalableaztreonam formulation to the lung endobronchial space of airways of apatient in need thereof, wherein the inhalable aztreonam formulationcomprises from about 1 to about 250 mg of aztreonam or apharmaceutically acceptable salt thereof per one dose, and thepharmaceutically acceptable salt thereof is selected from the groupconsisting of acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, bytyrate, camphorate, camphorsulfonate,citrate, digluconate, cyclopentanepropionate, dodecylsulfate,ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, lysinate, maleate,methanesulfonate, nicotinate,2-napthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate andundecanoate.
 23. The method of claim 22, wherein the pharmaceuticallyacceptable salt is lysinate.
 24. The method of claims 22 wherein saidgram-negative bacteria is Escherichia coli, Klebsiella pneumoniae,Klebsiella oxytoca, Pseudomonas aeruginosa, Haemophilus influenzae,Proteus mirabilis, Serratia marcescens, Burkholderia cepacia,Stenotrophomonas maltophilia, Alcaligenes xylosoxidans or multidrugresistant Pseudomonas aeruginosa.